Fatty acids are acyl lipids that are found in a variety of plant tissues, including the triacylglycerols in oil bodies of seeds and fruits, as well as the glycolipids and phospholipids in leaves, roots or shoots. Fatty acids include saturated and unsaturated monocarboxylic acids with unbranched even-numbered carbon chains, such as the unsaturated fatty acids: oleic (18:1, et al. a C18 chain with a double bond), linoleic (18:2) and linolenic (18:3) acid.
Plants may synthesize fatty acids in plastids using acetyl-CoA and malonyl-CoA as substrates. At least 30 enzymatic reactions may be involved in multiple cycles of condensation, reduction of the 3-keto group, dehydration and reduction of the double bond, leading to synthesis of palmitoyl-ACP (16:0-ACP) and stearoyl-ACP (18:0-ACP). A soluble enzyme, stearoyl-ACP Δ9 desaturase may introduce a first double bond in the conversion of the fatty acid stearoyl-ACP to oleoyl-ACP (18:1-ACP) (Shanklin and Somerville, Proc. Natl. Acad. Sci. USA 88: 2510-2514, 1991). Acyl-ACPs may be used for plastid lipid synthesis through transfer of free fatty acids from ACP to glycerol-3-phosphate or monoacylglycerol-3-phosphate. Alternatively, free fatty acids may be released from ACP by acyl-ACP thioesterases. These free fatty acids may be used to form acyl-CoAs and for the synthesis of other plant lipids including storage lipids in seeds. Further desaturation of fatty acids may be carried out by membrane bound desaturases of a chloroplast and endoplasmic reticulum.
In Arabidopsis, two loci, FAD2 and FAD3, have been shown to affect the desaturation of extraplastid lipids, which may lead to the synthesis of polyunsaturated fatty acids (Miquel and Browse, J. Bio. Chem. 267: 1502-1509, 1992.). Specifically, synthesis of the polyunsaturated fatty acids linoleic acid (Δ9,12-18:2) and α-linolenic acid (Δ9,12,15-18:3) may begin with the conversion of oleic acid (Δ9-18:1) to linoleic acid, the enzymatic step may be catalyzed by microsomal ω-6 oleic acid desaturase (FAD2). The linoleic acid may then be converted to α-linolenic acid through further desaturation by ω-3 linoleic acid desaturase (FAD3). Coding sequences of FAD2 genes have been reported in several Brassica species (Tanhuanpää et al., Mol. Breed. 4: 543-550, 1998; Singh et al., Plant Physiol. 109: 1498, 1995; Marillia and Taylor, Plant Physiol. 120: 339, 1999).
Mutational inactivation of a FAD2 gene has been reported in some species. For example, in cultured peanut, a mutation in the PFAD2-A gene coding sequences results in a non-functional protein (Jeong et al., Proceedings of the 3rd National Plant Lipid Cooperative Meeting, 1999. South Lake Tahole, Calif.). The Arabidopsis FAD2 mutant line FAD2-5, caused by T-DNA insertion into the FAD2 gene, shows increased oleic acid content (Okuley et al., Plant Cell 6: 147-158,1994).
Significant efforts have been made to manipulate the fatty acid profile of plants, particularly oil-seed varieties such as Brassica spp. that are used for the large-scale production of commercial fats and oils (see for example U.S. Pat. No. 5,625,130 issued to Grant et al. 29 Apr. 1997; U.S. Pat. No. 5,668,299 issued to DeBonte et al. 16 Sep. 1997; U.S. Pat. No. 5,840,946 issued to Wong et al. 24 Nov. 1998; U.S. Pat. No. 5,850,026 issued to DeBonte et al. 15 Dec. 1998; U.S. Pat. No. 5,861,187 issued to DeBonte et al. 19Jan. 1999; and U.S. Pat. No. 6,084,157 issued to DeBonte et al. 4 Jul. 2000).
An increase in the oleic acid content of plant oils may be desirable for some applications. For human consumption, vegetable oils rich in oleic acid may be considered superior products compared to oils rich in polyunsaturated fatty acids. The term “canola” has been used to describe varieties of Brassica spp. containing low erucic acid (Δ13-22:1) and low glucosinolates. For example, in the U.S., under 21 CFR 184.1555, low erucic acid rapeseed oil derived from Brassica napus or Brassica campestris is recognized as canola oil where it has an erucic acid content of no more than 2% of the component fatty acids (Table I sets out the Food Chemicals Codex (1996) specifications for canola oil). Plant breeders have also selected canola varieties that are low in glucosinolates, such as 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl or 2-hydroxy-4-pentenyl glucosinolate. Canola quality meal may for example be defined as having a glucosinolate content of less than 30 micromoles of aliphatic glucosinolates per gram of oil-free meal. Currently, the principle commercial canola crops comprise B. napus and B. rapa (campestris) varieties.
TABLE IFood Chemicals Codex (1996) Specifications for Canola OilPropertyCanola OilFatty Acids, % by weight<14<0.114:0 myristic<0.216:0 palmitic<6.016:1<1.018:0<2.518:1 oleic>50.018:2 linoleic<40.018:3 linolenic<14.020:0<1.020:1<2.022:0<0.522:1 crucic<2.024:0<0.224:1<0.2Acid value<6Cold TestPasses testColour (AOCS-Wesson)<1.5R/15YFree fatty acids (as oleic)<0.05%Heavy metals (as Pb)  ≦5 mg/kgIodine value110-126Lead<0.1 mg/kgPeroxide value≦10 meq/kgRefractive index1.465-1.467Saponifiable value178-193Stability≧7 hSulfur ≦10 mg/kgUnsaponifiable matter≦1.5%Water≦0.1%
B. juncea is an amphidiploid plant of the Brassica genera that is generally thought to have resulted from the hybridization of B. rapa and B. nigra. Under some growing conditions, B. juncea may have certain superior traits to B. napus and B. rapa. These superior traits may include higher yield, better drought and heat tolerance and better disease resistance. However, as a source of oils for human consumption, B. juncea is generally thought to have a less desirable fatty acid profile compared to the current canola crops. For example, the original wild type B. juncea varieties may contain low oleic acid (˜20%) and high erucic acid in the seed oil.
In the early 1980's, a low erucic acid B. juncea was reported (Kirk and Oram, J. Aust. Inst. Agric. Sci. 47: 51-52, 1981). However, this low erucic acid B. juncea also reportedly contains low oleic acid and high linoleic acid. Continued breeding efforts have focused on lowering the linoleic acid content and increasing the oleic acid content of B. juncea seed oil. Cosuppression strategies targeting FAD2 have for example recently been used to produce genetically modified B. napus and B. juncea varieties having elevated oleic acid content (Stotjesdijk et al., 1999). The relative unpredictability of cosuppression and antisense approaches may, however, detract from the usefulness of this approach in efficiently generating new varieties.
U.S. Pat. No. 6,303,849 issued to Potts et al. on 16 Oct. 2001 (incorporated herein by reference) discloses B. juncea lines having an edible oil that has properties similar to canola. The B. juncea lines disclosed therein have a lineage that includes B. juncea lines J90-3450 and J90-4316, deposited as ATCC Accession Nos 203389 and 203390 respectively (both of which were deposited by Agriculture and Agri-Food Canada under the terms of the Budapest Treaty on 23 Oct. 1998 at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. USA 20110-2209). There remains a need for novel varieties of B. juncea having favourable fatty acid profiles.